Six investigators seek support in this Program Project to explore the site- specific interactions of drugs, peptides and proteins with the key regulatory regions of HIV RNA using structural techniques. Understanding these processes on a molecular level will suggest new targets for the design of antiviral agents. a) The tat protein-TAR and rev protein-RRE interaction will be studied by X-ray crystallography and high resolution NMR. Structural models for these interactions will also be based on footprinting studies using chemical nucleases and the targeted scission of the regulatory regions by peptide linked to 1,10-phenanthroline-copper. b) Sequence-specific inhibitors of transcription and reverse transcriptase will be devised based on kinetic, chemical and crystallographic results. Their potential as antiviral agents will be explored if potent inhibition is observed. c) Transdominant rat mutants will be characterized by their interaction with TAR and the TAR binding cellular protein TRP-185 Dynamic 3D Profile Analysis of TRP-185 and computational structural studies of TAT and its derivatives will be carried. d) A single PCR-based assay for parasitic and viral etiological disease agents in blood will be devised to monitor the progression of adventitious infection in HIV patients. It will be based on a assay previously developed for Trypanosoma cruzi which relies on the nuclease activity of 1,10-phenanthroline-copper to cut DNA into amplified fragments. GRANT-=P01GM395580006 This proposal addresses the mechanisms by which mutant tat proteins inhibit wild-type tat proteins from activating HIV-1 gene expression. Our laboratory demonstrated that so called dominant negative or transdominant tat mutants can be constructed by introducing substitutions or truncations into the basic domain of tat. Several of these mutants are able to inhibit wild-type tat function when both proteins are present in equimolar concentrations. Thus, it is possible that such mutants may have therapeutic potential in the treatment of HIV-1 infection. However it will be critical to determine the mechanism of this inhibition both the understand tat function and to develop better inhibitors of tat function. This proposal has as its specific aims: (1) to create additional transdominant tat mutants (2) to construct recombinant HIV-1 viruses that contain transdominant tat mutants (3) to produce wild-type and mutant tat proteins in vaccinia virus and bacterial expression systems to test their ability to alter HIV-1 gene expression (4) to determine the ability of cellular proteins to associate with wild-type and transdominant tat proteins. These studies should lead to a better understanding of tat function and provide the basis for the development of inhibitors of tat function.
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